Presence Of DPC Micelles Research Articles

Molecular basis of the anticancer, apoptotic and antibacterial activities of Bombyx mori Cecropin A

As Cecropin XJ, Cecropin A from Bombyx mori is one of the very few antimicrobial peptides having shown activity against esophageal cancer cells. It displays remarkable sequence-similarity to Cecropin XJ but slightly enhanced activity. In this work we show by NMR that both peptides are unstructured in solution but get structured in the presence of DPC micelles, mimicking the surface of biological membranes. In order to get insight into the molecular basis of its anticancer, antimicrobial and antifungal activity, we have investigated by MD simulations their interaction with a large variety of lipid bilayers mimicking cancer, mitochondrial, bacterial and fungal membranes. At variance with CecXJ, organized in two main helices, CecA tends to form a three helix bundle resulting in enhanced adaptability to its membrane targets. A specificity for the headgroup of phosphatidylserine and affinity for phosphatidylglycerol and cardiolipin may account for its selective targeting of cancer, bacterial and mitochondrial membranes, respectively.

The impact of phosphatidylserine exposure on cancer cell membranes on the activity of the anticancer peptide HB43.

HB43 (FAKLLAKLAKKLL) is a synthetic peptide active against cell lines derived from breast, colon, melanoma, lung, prostate, and cervical cancers. Despite its remarkable spectrum of activity, the mechanism of action at the molecular level has never been investigated, preventing further optimization of its selectivity. The alternation of charged and hydrophobic residues suggests amphipathicity, but the formation of alpha-helical structure seems discouraged by its short length and the large number of positively charged residues. Using different biophysical and in silico approaches we show that HB43 is completely unstructured in solution but assumes alpha-helical conformation in the presence of DPC micelles and liposomes exposing phosphatidylserine (PS) used as mimics of cancer cell membranes. Membrane permeabilization assays demonstrate that the interaction leads to the preferential destabilization of PS-containing vesicles with respect to PC-containing ones, here used as noncancerous cell mimics. ssNMR reveals that HB43 is able to fluidify the internal structure of cancer-cell mimicking liposomes while MD simulations show its internalization in such bilayers. This is achieved by the formation of specific interactions between the lysine side chains and the carboxylate group of phosphatidylserine and/or the phosphate oxygen atoms of targeted phospholipids, which could catalyze the formation of the alpha helix required for internalization. With the aim of better understanding the peptide biocompatibility and the additional antibacterial activity, the interaction with noncancerous cell mimicking liposomes exposing phosphatidylcholine (PC) and bacterial mimicking bilayers exposing phosphatidylglycerol (PG) is also described.

Antimicrobial peptide protegrin-3 adopt an antiparallel dimer in the presence of DPC micelles: a high-resolution NMR study

A tendency to dimerize in the presence of lipids was found for the protegrin. The dimer formation by the protegrin-1 (PG-1) is the first step for further oligomeric membrane pore formation. Generally there are two distinct model of PG-1 dimerization in either a parallel or antiparallel β-sheet. But despite the wealth of data available today, protegrin dimer structure and pore formation is still not completely understood. In order to investigate a more detailed dimerization process of PG-1 and if it will be the same for another type of protegrins, in this work we used a high-resolution NMR spectroscopy for structure determination of protegrin-3 (RGGGL-CYCRR-RFCVC-VGR) in the presence of perdeuterated DPC micelles and demonstrate that PG-3 forms an antiparallel NCCN dimer with a possible association of these dimers. This structural study complements previously published solution, solid state and computational studies of PG-1 in various environments and validate the potential of mean force simulations of PG-1 dimers and association of dimers to form octameric or decameric β-barrels.

Engineering the Endothelin A and B Receptors using a Soluble Template for Structural Analysis and Small Molecule Screening

With almost 1000 unique G Protein Coupled Receptors (GPCR's) mapped in the human genome, and an estimated 40% of current marketed drugs targeting these proteins, there is little question that it worthwhile to develop new methods to more efficiently screen for small molecule agonists and antagonists. One such group of GPCRs, the endothelin receptors, is home to one of the most potent vasoconstrictors, the Endothelin A receptor (ETA) which is an excellent target for selective antagonists for the treatment of pulmonary arterial hypertension. We have developed a method for screening selective antagonists which target intracellular domains of ETA and ETB using a soluble template (to mimic a region of the transmembrane domains of ETA and ETB) fused to the IC3 loop and Carboxyl terminus of ETA and ETB. Additionally, we have generated a structural model of the ETA Carboxyl terminus (Residues 373-427) from NMR data both in solution and in the presence of DPC micelles. We hope that with these tools we can soon identify and develop targeted inhibitors of vasoconstriction through high throughput screening and rational design.

Nmr Structural Studies of Antimicrobial Peptides, LPcin

Lactophoricin (LPcin-I) is an antimicrobial, amphiphatic, cationic peptide with 23-amino acid residues isolated from bovine milk. Its analogous peptide, LPcin-II, lacks six N-terminal amino acids compared to LPcin-I. Interestingly, LPcin-II does not display any antimicrobial activity, whereas LPcin-I inhibits the growth of both Gram-negative and Gram-positive bacteria without exhibiting any hemolytic activity. Uniformly 15N-labeled LPcin peptides were prepared by the recombinant expression of fusion proteins in E. coli and their properties were characterized by mass spectrometry (MS), Circular dichroism (CD) spectroscopy, and antimicrobial activity tests. To understand the structure-activity relationship of these two peptides, they were studied in model membrane environments by a combination of solution and solid-state NMR spectroscopy. We determined the tertiary structure of LPcin-I and LPcin-II in the presence of DPC micelles by solution NMR spectroscopy. Magnetically aligned unflipped bicelle samples were used to investigate the structure and topology of LPcin-I and LPcin-II by solid-state NMR spectroscopy. We will also discuss about the LPCin-Analogs that have much more powerful antimicrobial activity and 800MHz home-built solid-state NMR probe for bilayers.[1] Protein Expression and Purification 65 (2009), 23 [2] Biophysical J 101 (2011) 1193.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Structural flexibility and the positive charges are the key factors in bacterial cell selectivity and membrane penetration of peptoid-substituted analog of Piscidin 1

Piscidin 1 (Pis-1) is a novel cytotoxic peptide with a cationic α-helical structure isolated from the mast cells of hybrid striped bass. In our previous study, we showed that Pis-1[PG] with a substitution of Pro 8 for Gly 8 in Pis-1 had higher bacterial cell selectivity than Pis-1. We designed peptoid residue-substituted peptide, Pis-1[NkG], in which Gly 8 of Pis-1 was replaced with Nlys (Lys peptoid residue). Pis-1[NkG] had higher antibacterial activity and lower cytotoxicity against mammalian cells than Pis-1 and Pis-1[PG]. We determined the tertiary structure of Pis-1[PG] and Pis-1[NkG] in the presence of DPC micelles by NMR spectroscopy. Both peptides had a three-turn helix in the C-terminal region and a bent structure in the center. Pis-1[PG] has a rigid bent structure at Pro 8 whereas Pis-1[NkG] existed as a dynamic equilibrium of two conformers with a flexible hinge structure at Nlys 8. Depolarization of the membrane potential of Staphylococcus aureus and confocal laser-scanning microscopy study revealed that Pis-1[NkG] effectively penetrated the bacterial cell membrane and accumulated in the cytoplasm, whereas Pis-1[PG] did not penetrate the membrane but remained outside or on the cell surface. Introduction of a lysine peptoid at position 8 of Pis-1 provided conformational flexibility and increased the positive charge at the hinge region; both factors facilitated penetration of the bacterial cell membrane and conferred bacterial cell selectivity on Pis-1[NkG].

Improving Metabolic Stability by Glycosylation: Bifunctional Peptide Derivatives That Are Opioid Receptor Agonists and Neurokinin 1 Receptor Antagonists

In order to obtain a metabolically more stable analgesic peptide derivative, O-beta-glycosylated serine (Ser(Glc)) was introduced into TY027 (Tyr-d-Ala-Gly-Phe-Met-Pro-Leu-Trp-NH-3',5'-Bzl(CF(3))(2)) which was a previously reported bifunctional compound with delta/micro opioid agonist and neurokinin-1 receptor antagonist activities and with a half-life of 4.8 h in rat plasma. Incorporation of Ser(Glc) into various positions of TY027 gave analogues with variable bioactivities. Analogue 6 (Tyr-d-Ala-Gly-Phe-Nle-Pro-Leu-Ser(Glc)-Trp-NH-3',5'-Bzl(CF(3))(2)) was found to have effective bifunctional activities with a well-defined conformation with two beta-turns based on the NMR conformational analysis in the presence of DPC micelles. In addition, 6 showed significant improvement in its metabolic stability (70 + or - 9% of 6 was intact after 24 h incubation in rat plasma). This improved metabolic stability, along with its effective and delta selective bifunctional activities, suggests that 6 could be an interesting research tool and possibly a promising candidate as a novel analgesic drug.

The Micelle-Associated 3D Structures of Boc-Y(SO3)-Nle-G-W-Nle-D-2-phenylethylester (JMV-180) and CCK-8(s) Share Conformational Elements of a Calculated CCK1Receptor-Bound Model

JMV-180 ( 1) and CCK-8(s) are high affinity ligands at the CCK 1 receptor that have similar and different actions via this receptor. Here we calculate the tertiary structure of 1 or CCK-8(s) in the presence of dodecylphosphocholine micelles at pH 5.0 and 35 degrees C from 2D (1)H NMR data recorded at 600 MHz. The NMR derived 3D structures of 1 and CCK-8(s) share a common type I beta-turn around residues Nle3/M3 and G4 and diverge from each other structurally at the N- and C-termini. The fluorescence and circular dichroism spectral properties of these peptides are consistent with their NMR derived structures. The structures determined in the presence of DPC micelles are compared to available models of 1 or CCK-8(s) bound to the CCK 1 receptor. For CCK and 1, these comparisons show that DPC micelle associated structures duplicate some important aspects of the models calculated from cross-linking derived constraints at the CCK 1 receptor.

Three-dimensional structure of the two peptides that constitute the two-peptide bacteriocin plantaricin EF

The three-dimensional structures of the two peptides plantaricin E (plnE; 33 residues) and plantaricin F (plnF; 34 residues) constituting the two-peptide bacteriocin plantaricin EF (plnEF) have been determined by nuclear magnetic resonance (NMR) spectroscopy in the presence of DPC micelles. PlnE has an N-terminal α-helix (residues 10–21), and a C-terminal α-helix-like structure (residues 25–31). PlnF has a long central α-helix (residues 7–32) with a kink of 38 ± 7 degrees at Pro20. There is some flexibility in the helix in the kink region. Both helices in plnE are amphiphilic, while the helix in plnF is polar in its N-terminal half and amphiphilic in its C-terminal half. The α-helical content obtained by NMR spectroscopy is in agreement with CD studies. PlnE has two GxxxG motifs which are putative helix–helix interaction motifs, one at residues 5 to 9 and one at residues 20 to 24, while plnF has one such motif at residues 30 to 34. The peptides are flexible in these GxxxG regions. It is suggested that the two peptides lie parallel in a staggered fashion relative to each other and interact through helix–helix interactions involving the GxxxG motifs.

Three-dimensional structure of the two peptides that constitute the two-peptide bacteriocin lactococcin G

The three-dimensional structures of the two peptides, lactococcin G-α (LcnG-α; contains 39 residues) and lactococcin G-β (LcnG-β, contains 35 residues), that constitute the two-peptide bacteriocin lactococcin G (LcnG) have been determined by nuclear magnetic resonance (NMR) spectroscopy in the presence of DPC micelles and TFE. In DPC, LcnG-α has an N-terminal α-helix (residues 3–21) that contains a GxxxG helix–helix interaction motif (residues 7–11) and a less well defined C-terminal α-helix (residues 24–34), and in between (residues 18–22) there is a second somewhat flexible GxxxG-motif. Its structure in TFE was similar. In DPC, LcnG-β has an N-terminal α-helix (residues 6–19). The region from residues 20 to 35, which also contains a flexible GxxxG-motif (residues 18–22), appeared to be fairly unstructured in DPC. In the presence of TFE, however, the region between and including residues 23 and 32 formed a well defined α-helix. The N-terminal helix between and including residues 6 and 19 seen in the presence of DPC, was broken at residues 8 and 9 in the presence of TFE. The N-terminal helices, both in LcnG-α and -β, are amphiphilic. We postulate that LcnG-α and -β have a parallel orientation and interact through helix–helix interactions involving the first GxxxG (residues 7–11) motif in LcnG-α and the one (residues 18–22) in LcnG-β, and that they thus lie in a staggered fashion relative to each other.

The role of segment 32-47 of cholecystokinin receptor type A in CCK8 binding: synthesis, nuclear magnetic resonance, circular dichroism and fluorescence studies.

The segment 32-47 of the N-terminal extracellular domain of the type A cholecystokinn receptor, CCK(A)-R(32-47), was synthesized and structurally characterized in a membrane mimicking environment by CD, NMR and molecular dynamics calculations. The region of CCK(A)-R(32-47) encompassing residues 39-46 adopted a well-defined secondary structure in the presence of DPC micelles, whereas the conformation of the N-terminal region (segment 32-37) could not be uniquely defined by the NOE derived distance constraints because of local flexibility. The conformation of the binding domain of CCK(A)-R(32-47) was different from that found for the Intact N-terminal receptor tail, CCK(A)-R(1-47). To assess whether CCK(A)-R(32-47) was still able to bind the nonsulfated cholecystokinin C-terminal octapeptide, CCK8, a series of titrations was carried out in SDS and DPC micelles, and the binding interaction was followed by fluorescence spectroscopy. These titrations gave no evidence for complex formation, whereas a high binding affinity was found between CCK(A)-R(1-47) and CCK8. The different affinities for the ligand shown by CCK(A)-R(32-47) and CCK(A)-R(1-47) were paralleled by different interaction modes between the receptor segments and the micelles.The interaction of CCK(A)-R(32-47) with DPC micelles was much weaker than that of CCK(A)-R(1-47), because the former receptor segment lacks proper stabilizing contacts with the micelle surface. In the case of SDS micelles CCK(A)-R(32-47] was found to form non-micellar adducts with the detergent that prevented the onset of a functionally significant Interaction between the receptor segment and the micelle. It is concluded that tertiary structure interactions brought about by the 1-31 segment play a key role in the stabilization of the membrane bound, biologically active conformation of the N-terminal extracellular tail of the CCKA receptor.

The Alignment of a Voltage-Sensing Peptide in Dodecylphosphocholine Micelles and in Oriented Lipid Bilayers by Nuclear Magnetic Resonance and Molecular Modeling

The S4 segments of voltage-gated sodium channels are important parts of the voltage-sensing elements of these proteins. Furthermore, the addition of the isolated S4 polypeptide to planar lipid bilayers results in stepwise increases of ion conductivity. In order to gain insight into the mechanisms of pore formation by amphipathic peptides, the structure and orientation of the S4 segment of the first internal repeat of the rat brain II sodium channel was investigated in the presence of DPC micelles by multidimensional solution NMR spectroscopy and solid-state NMR spectroscopy on oriented phospholipid bilayers. Both the anisotropic chemical shift observed by proton-decoupled 15N solid-state NMR spectroscopy and the attenuating effects of DOXYL-stearates on TOCSY crosspeak intensities of micelle-associated S4 indicate that the central α-helical portion of this peptide is oriented approximately parallel to the membrane surface. Simulated annealing and molecular dynamics calculations of the peptide in a biphasic tetrachloromethane-water environment indicate that the peptide α-helix extends over ∼12 residues. A less regular structure further toward the C-terminus allows for the hydrophobic residues of this part of the peptide to be positioned in the tetrachloromethane environment. The implications for possible pore-forming mechanisms are discussed.

Oligomerization of protegrin-1 in the presence of DPC micelles. A proton high-resolution NMR study

Protegrins are members of a family of five Cys-rich naturally occurring cationic antimicrobial peptides. The NMR solution structure of protegrin-1 (PG-1) has been previously determined as a monomeric β-hairpin both in water and in dimethylsulfoxide solution. Protegrins are bactericidal peptides but their mechanism of action is still unknown. In order to investigate the structural basis of their cytotoxicity, we studied the effect of lipid micelles on the structure of PG-1. The NMR study reported in the present work indicates that PG-1 adopts a dimeric structure when it binds to dodecylphosphocholine micelles. Moreover, the amide proton exchange study suggests the possibility of an association between several dimers.